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Failure of dimethyl sulfoxide (DMSO) to alter thyroid function in the Sprague-Dawley rat
TOXICOLOGYAND
APPLIEDPHARMACOLOGY-,
Failure of Dimethyl Thyroid Function
73-80(1973)
Sulfoxide (DMSO) in the Sprague-Dawley
to Alter Rat1
M. GOLDMAN Department of Biology, University of South Dakota, Vermillion, South Dakota 57069 Received February 23,1972
Failure of Dimethyl Sulfoxide (DMSO) to Alter Thyroid Function in the Rat. GOLDMAN, M. (1973). Toxicol. Appl. Pharmacol. 24, 73-80. Male Sprague-Dawleyrats 60 days and 1 year of age were injected with 63% or 85% dimethyl sulfoxide (DMSO) and severalphasesof thyroid function examined. Iodide transport was not affected by injection of either 63‘A or 85‘A DMSO. There was no significant differencebetween the saline-injectedand DMSO-injected groupsin the thyroid :serumradioiodide concentration ratios (T:S). The responseof the thyroid gland to the injection of an acute substantial iodide load was unaltered by prior injection of 85% DMSO. The inhibition in glandularbinding of iodide was similar in both saline-injectedand DMSO-injected groups as evidenced by the thyroidal accumulationof newly formed organiciodineand thyroidal concentration of iodide. Chromatographic analysesof thyroid pronase hydrolyzates in saline-injectedand DMSO-injected rats revealeda similar reduction in labelingof iodothyronines,an increasein the fraction of labeled iodide, anincreasein thyroidal labelingof MIT with consequentelevation in ratio of [r311]MIT:DIT. The radioiodine releaserate wasalsonot affected by injection of DMSO or dermal application of DMSO. Presentresults indicate that DMSO was not effective in influencing thyroid function in the rat. Numerous laboratory experimentation and clinical investigations have revealed that dimethyl sulfoxide (DMSO) has a wide spectrum of biological activity and yet is remarkably nontoxic (Rosenkrantz et al., 1963). It has been reported that DMSO increases the permeability of biological membranes (Jacob et al., 1964), is a good solvent for many steroids(Kligman, 1965; Stoughton, 1965),and augmentspercutaneous penetration of various drugs (Horita and Weber, 1964; Stoughton and Fritsch, 1964). DMSO is also radioprotective against ionizing radiation (Ashwood-Smith, 1961), protects cellsand tissuesagainst freezing damage(Lovelock and Bishop, 1959),prevents the inflammatory response (Ward et al., 1967), and relieves pain due to headache (Ogden, 1967). A recent report indicates that DMSO may have an inhibitory action on thyroid function (Hagemarm and Evans, 1968). These investigators reported that DMSO inhibited 1311uptake by mice thyroid glands both in vivo and in vitro. The inhibitory effect of DMSO on 1311uptake in vitro was simply removed by washing the thyroid glands in saline. Radioiodine uptake curves obtained over the period oftime during which thyroid glands were incubated in 15‘A DMSO appeared to parallel those uptake 1 Supportedby a grant from the University of SouthDakota’sGeneralResearch/NASAFund. Copyright All rights
0 1973 by Academic Press, Inc. of reproduction in any form reserved.
73
74
GOLDMAN
curves obtained when thyroid glands were incubated in thiocyanate (to block active transport of iodide) plus propylthiouracil (to block organification of iodide). Moreover, injection of 63 ‘A DMSO in mice resulted in a slight but significant depression in 1311 uptake 1 hr after injection. However, the depression in radioiodine uptake was not observable in these mice 6 hr later, presumably due to the fall in DMSO blood content. Consequently, Hagemann and Evans (1968) suggested that DMSO inhibited both active transport and organification of iodine by the thyroid gland. The present study reports our findings in investigating the action of DMSO on iodide transport and organification of iodine by the thyroid gland in the rat in an attempt to elucidate the mechanism of action by which DMSO inhibits thyroid function.
METHODS Two groups of male Sprague-Dawley rats obtained from our colony aged 60 days and 1 year old were used in this study. The animals were maintained on Purina Laboratory Chow and water ad libitum. DMSO’ was used at 2 dosages, 63% and 85% (v/v in physiological saline). All control animals were injected ip with 0.5 ml of physiological saline. Thyroid:serum radioiodide concentration ratios (T:S). Rats were injected ip with 10 mg methimazole3 in physiological saline in order to block organification of iodine, followed 1 hr later by an ip injection of 3-5 $i of carrier-free 13114.The administration of the radioiodide was preceded by a single ip injection of 0.5 ml DMSO in a concentration of 63 % or 85 % 5 min before the injection of the 1311.One hour later the thyroid glands were removed under light ether anesthesia, dissected, carefully weighed, and homogenized in 0.5 ml NaClaTris buffer, pH 8.5. An aliquot of homogenate was counted in a Nuclear Chicago well-type scintillation counter in order to calculate the amount of radioactivity in 1 g of thyroid tissue. Blood was obtained from the dorsal aorta after removal of the thyroid gland and an aliquot of serum counted to determine the radioactivity in 1 ml of serum. The thyroid-serum radioiodide concentration ratio (T:S) was calculated by determining the ratio of radioiodide per gram of thyroid to that of the radioiodide per milliliter of serum (Rosenberg et al., 1964). Distribution of 1311in hydrolyzed thyroidglands. Rats (60 days of age) received an ip injection of 0.5 ml 85 % DMSO which was followed 5 min later by an ip injection of 10 &i 1311together with 200 pg carrier iodide. The thyroid glands were removed 1 hr later, homogenized immediately in the NaCI*Tris buffer, pH 8.5, and hydrolyzed with pronase5. The radioactivity in an aliquot of homogenate was counted to determine the radioiodine uptake. The iodoaminoacids of the hydrolyzate were fractionated by paper chromatography in a collidine-3 N ammonia (3 : 1 v/v) solvent system and the position of the bands of radioactivity containing the iodinated amino acids were determined by autoradiography of the chromatograms. The radioactivity in these bands was determined after removal of the radioactive strips from the chromatogram and placement in glass vials following the procedure of Rosenberg et al. (1964). * Obtained from Mallinckrodt, St. Louis, Missouri. 3 I-Methyl-2-mercaptoimidazole, a gift from Eli Lilly and Company, Indianapolis, Indiana. 4 N.T obtained from Amersham/Searle, Arlington Heights, Illinois. .5 Streptomyces griseus protease, a product of Calbiochem, Los Angeles, California,
DMSO
AND
THYROID
FUNCTION
75
Distribution of 1311 in unhydrolyzed thyroid glands. Thyroid glands from another group of rats (60 days of age) similarly injected ip with 85 % DMSO followed 5 min later by an ip injection of 20 &i 131Ttogether with 200 ,ug iodide were homogenized in the NaClaTris buffer pH 8.5, and substantial aliquots of the unhydrolyzed glands were chromatographed using the same solvent system as above. Autoradiographs of the chromatograms localized the radioactivity into 2 bands: a band at the origin assumed to represent organic iodine and a band of iodide. The concentration of newly formed protein bound iodine (PB 12’1) and iodide 12’1 were calculated from the specific activity of the injected iodide, 1311uptake, and the fraction of 1311at the originaccording to the procedure of Rosenfeld and Rosenberg (I 966). Thyroid radioiodine release rate. Thyroidal 1311 release was measured by external neck counts taken over the thyroid region twice daily. The counting table had a top consisting of a lead plate 2 inches thick with an opening 1 inch in diameter directly above a scintillation probe which had a 2-inch NaI crystal. The probe was connected to a Nuclear Chicago scaler. Rats lightly anesthetized with ether were placed on the counting table and the neck positioned over the opening until the thyroid region was precisely above the opening over the scintillation probe. The initial thyroid count was taken 36 hr after an ip injection of 20 pCi carrier-free 1311and was considered as 100 )‘,; of thyroidal L31l. All subsequent counts were expressed as a percentage of the initial thyroidal uptake and plotted as a function of time on semilog paper. Blockage of thyroidal reaccumulation of 1311liberated from degradation of endogeneously labeled iodinated amino acids was not attempted. The procedure used was based on the method described by Albert (1951) and Reineke and Singh (1955). Hair from the lateral abdominal area was removed by plucking from 5 male rats 60 days of age, and during determination of the thyroidal 1311release rate 0.1 ml 1000; DMSO was dermally applied daily by means of a cahbrated medicine dropper. In a second experiment, after a straight line of more than 5 individual points was obtained for the thyroidal 1311release curve, 5 male rats 1 year old were injected ip daily with 0.5 ml 85 % DMSO. Analysis of the significance of differences between groups was done by means of Student’s t test. Ap value co.01 was considered statistically significant. RESULTS
Table 1 shows the data obtained in 2 experiments in which 2 different doses of DMSO were utilized in examining the effects of DMSO on the thyroid: serum radioiodide concentration ratio (T: S). The T: S values for the 2 control groups are typical of rats of that age group in our colony. Since 63 % DMSO did not affect the ability of the thyroid to trap iodide, the dose was consequently increased to 85 % DMSO. Again, the injected 85 % DMSO had no significant effect on iodide transport. The T: S ratios in the 2 experiments were not significantly different in the saline-injected and DMSUinjected rats. Thyroidal Organl$cation
of Iodide
The capacity to organify iodine can be studied by examining the thyroidal content of newly accumulated organic iodine after injection of an iodide load (Rosenfeld and
76
GOLDMAN
TABLE EFFECT OF 63 % DMSO
Group
No. of rats
Control DMSO (63 %) Control
10
DMSO
(83 %)
9 12 11
1
AND 85 % DMSO
Body weight (d
Age 60 days 60 days 1 year 1 year
ON T: S IODIDE IN MALE RATS’
201 * 8.3 183 rt 11.0 400 f 12.1 389 h 14.0
Thyroid weight @x4 13.0 zt 0.8 15.2 zt 0.7 17.7 i 1.1 16.1 zt 1.3
T:S 22.7 18.4 48.0 42.8
It rt k k
2.1b 2.6” 3.1 3.6”
a Rats received a single ip injection of 0.5 ml DMSO (63 % or 85 %) 5 min before ip injection of r3rI; thyroid glands removed 1 hr later and thyroid radioiodide concentration/serum radioiodide concentration ratio calculated. b Mean value * SE. c Not significantly different from controls.
Rosenberg, 1966). Table 2 shows the effect of 200 pg of iodide in causing a reduction in organic binding of iodine by the thyroid gland (Wolff and Chaikoff, 1948) in salineinjected and 85% DMSO-injected rats. The data presented in Table 2 show that the 1311uptake, the fraction of 1311organically bound, the accumulation of newly formed organic iodine, and the concentration of accumulated inorganic iodide were not significantly different in the thyroid glands of saline-injected and DMSO-injected rats subjected to an acute iodide load. Alterations in intrathyroidal iodine metabolism which occurred in response to the injection of 200 pg iodide were not influenced by DMSO. TABLE
2
EFFECT OF 85 % DMSO ON ORGANIFICATION OF AN INJECTED IODIDE LOAD (200 pg) IN MALE RATS 60 DAYS OLDO Organic
1311Uptake Group
No. of rats
Control DMSO (85 %)
12 12
1311
‘A injected
organically
(dosehw)
bound
0.01 f 0 0.01 i 0
11.1 f 3.8
12.7 zt 3.0
Iodine newly formed (wims) 2.30 zk 0.52 1.59 rt 0.59
Inorganic iodide accumulated Cx/mg) 15.8 4 1.7b 13.4 f 1.7
a Rats received a single ip injection of 0.5 ml 85 % DMSO 5 min before ip injection of 20 &i r3’I plus 200 pg carrier iodide; thyroid glands removed 1 hr later, homogenized, and chromatographed. b Mean value f SE.
Radioiodine Distribution in Componentsof Pronase Thyroid Hydrolyzates
’ Chromatographic analyses of thyroid hydrolyzates are shown in Table 3. A marked reduction in percentage of 1311in iodothyronines (T3 + T4) and iodotyrosines (MIT and DIT) is observable in both saline-injected and 85 ‘A DMSO-injected rats. The large increase in the fraction of labeled iodide occurred in both groups of rats and again there was no significant difference in percentage of thyroidal label in this component. These results are consistent with the data observed in Table 2, showing the marked
DMSO
AND
THYROID
77
FUNCTION
impairment in thyroidal capacity to bind iodine organically after injection of 200 pg iodide. The fraction of thyroidal label in MIT is considerably higher than in DIT in both groups of rats and results in a significant increase in MIT: DIT ratio (Table 3). The data shown in Tables 2 and 3 are evidence that the Wolff-Chaikoff effect (inhibition of iodothyronine synthesis) occurred in both saline-treated and DMSO-treated rats in response to the injected iodide load without any influence of DMSO on the iodide block in organification of iodine. TABLE DISTRIBUTION OF RATS 60 DAYS
Group Control DMSO
OF 1311(‘A) IN CHROMATOGRAMS OF AGE INJECTED WITH 85 ‘A IODIDP
No. of rats 10
(85 %)
10
3 OF THYROID HYDROLYZATES AND 200 pg OF CARRIER
DMSO
Origin
MIT
DIT
0.7 iz 0.2 0.5 ~0.2
3.0 50.8 6.7 * 1.6
1.5 i0.6 3.6 zk 1.7
Tx+Tb 1.5 ho.6 1.7 ztO.4
1311 93.4 -+ 1.8 87.5 312.8
MIT:DIT 2.0 ~~0.5~ 1.0 $I 0.5
” Rats received a single ip injection of 0.5 ml 85 % DMSO 5 min before ip injection of 20 &i 13’1 plus 200 pg carrier iodide; thyroid glands removed 1 hr later, homogenized, hydrolyzed with pronase and chromatographed. MIT; monoiodotyrosine; DIT, Diiodotyrosine; TA, triiodothyronine; Tj, thyroxine. h Mean value -I SE.
Tllyroidal Radioiodine Release
Figure 1 shows the effect of DMSO on the thyroidal secretion rate. Daily in,jections of 0.5 ml 85 y{ DMSO did not alter the rate of 1311secretion from the thyroid gland. Dermal application of concentrated DMSO also did not alter 1311 release from the
0
I
I
I
I
30
60
90
120
I
150
HOURS FIG. 1. Effect of DMSO on thyroidal radioiodine release, Male rats 60 days of age were injected ip daily with 0.5 ml 85 % DMSO where noted (4). The curve represents the means f SE of determinations made on 5 rats.
78
GOLDMAN
thyroid. Since there were no changes in slope of the thyroid 1311release rates when DMSO was injected or dermally applied, only the thyroidal radioiodine release curve for DMSO injected rats is shown. The biological half-life determined graphically was 3.3 days for both control and DMSO-treated rats. DISCUSSION The active transport of iodide, the first step in the metabolism of iodine, can be examined independently and separately of subsequent steps in thyroid hormone biosynthesis. The efficiency of the thyroid gland in iodide transport is measured experimentally as the thyroid : serum radioiodide concentration ratio (T: S) and expresses the concentration gradient for radioiodide which the thyroid maintains against the plasma (Vanderlaan and Vanderlaan, 1947). The data presented in this study of the ineffectiveness of DMSO in depressing the active transport of iodide are at variance with the suggestion of Hagemann and Evans (1968) that DMSO inhibits this phase of thyroid function. DMSO given at 2 different doses did not inhibit iodide transport by the thyroid gland. Synthesis of the thyroid hormones, thyroxine and triiodothyronine, results from the coupling of iodotyrosines subsequent to organic binding of the trapped iodide. Hagemann and Evans (1968) claim that DMSO also inhibits organification of iodine by the thyroid gland. While administration of small doses of carrier iodide leads to an increase in thyroidal organic iodinations, large doses of iodide result in a depression of organic binding of iodide. This temporary inhibitory effect on organification of iodide and consequent depression in synthesis of thyroid hormones as a result of a large dose of iodide has been termed the Wolff-Chaikoff effect and has been shown to be related to the increased concentration of intrathyroidal iodide (Raben, 1949). Consequently, an estimate of the capacity of the thyroid gland to organify iodine can be obtained by examining the glandular content of newly formed organic iodine after injection of a substantial iodide load (Deodhar and Rosenberg, 1969). Our results do not confirm Hagemann and Evans’ (1968) suggestion that DMSO inhibits thyroidal organification of iodide. The organic iodine and iodide newly accumulated in the thyroid glands of control and DMSO injected rats were similar after injection of the 200 pg iodide. The inhibition in glandular binding of iodide by the injected iodide was of similar magnitude in both control and DMSO-treated rats and was not influenced by injection of DMSO. The marked depression in thyroidal labeling of iodothyronines, iodotyrosines and the increased fraction of 1311-labeled iodide were not significantly different in the 2 groups. Moreover, the increase in labeling of the MIT component was reflected as a significant increase in MIT:DIT ratio in both groups. Galton and Pitt-Rivers (1959) have pointed out that the MIT : DIT ratio is very high when organic binding of iodide is most depressed. No inhibition in thyroidal secretion rate was observed after dermal application of an injection of 85 7; DMSO. In contrast to the demonstration of an inhibitory effect of DMSO on thyroid function in the mouse reported by Hagemann and Evans (1968), the present investigation does not reveal any demonstrable effect of DMSO on thyroid function in the rat. Conse-
DMSO AND THYROID FUNCTION
79
quently, we have not been able to confirm the mechanism of action for DMSO inhibition of thyroid function suggestedby theseinvestigators. Although a remarkable lack of speciesvariation in responseto DMSO has been reported (Smith et al., 1967), our results do not deny the existence of possibledifferences in metabolism of DMSO in the rat and mousewhich could resolve the disparity in findings reported here with those of Hagemann and Evans (1968), particularly in view of the reports of speciesdifferences in thyroid function in the rat and mouse (Wollman and Reed, 1959; Silverstein and Bates, 1961). REFERENCES ALBERT,
A. (1951).The in vivo determinationof the biological decayof thyroidal radioiodine.
Endocrinology
48, 334-338.
M. J. (1961).The radioprotective action of dimethyl sulphoxideand various other sulphoxides.Int. J. Radiat. Biol. 3, 41-48. DEODHAR, S. D. and ROSENBERG, I. N. (1969). Effects of actinomycin D on the thyroid. ASHWOOD-SMITH,
Endocrinology
85, 629-637.
R. (1959).The effect of excessiveiodine on the thyroid of the rat. Endocrinology 64, 835-839. HAGEMANN, R. F. and EVANS, T. C. (1968). Reversibleinhibition of thyroidal uptake of i311by dimethyl sulfoxideProc. Sot. Exp. Biol. Med. 128, 1008-1010. HORITA, A. and WEBER, J. (1964). Skin penetrating property of drugs dissolvedin dimethyl sulfoxide (DMSO) and other vehiclesLife Sci. 3, 1389-1395. JACOB, S. W., BISCHEL, M. and HERSCHLER, R. J. (1964). Dimethyl sulfoxide: effectson the permeability of biologic membranes.(Preliminary Report.) Curr. Ther. Rex 6, 193-198. KLIGMAN, A. M. (1965). Topical pharmacology and toxicology of dimethyl sulfoxide. I. GALTON,
V. A. and PITT-RIVERS,
J. Amer. Med. Ass. 193, 796-804. LOVELOCK, J. E. and BISHOP, M. W. H. (1959).Prevention of freezing damage to living cells by dimethyl sulphoxide. Nature (London) 183, 13941395. OGDEN, H. D. (1967).Experience with DMSO in treatment of headache. Ann. N. Y. Acad. Sci. 141,646-648. RABEN, M. S. (1949).The paradoxical effects of thiocyanate and of thyrotropin on the organic binding of iodine by the thyroid in the presence of large amounts of iodide. Endocrino1og.v 45,296-304. REINEKE, E. P. and SINGH, 0. N. (1955). Estimation of thyroid hormone secretion rate of intact rat. Proc. Sot. Exp. Biol. Med. g&203-207. ROSENBERG, L. L., GOLDMAN, M., LA ROCHE, G. and DIMICK, W. K. (1964).Thyroid function in rats and chickens: equilibrium of injected iodide with existing thyroidal iodine in LongEvans rats and white Leghorn chickens.Endocrinology 74,212-225. ROSENFELD, P. S. and ROSENBERG, I. N. (1966).Early enhancement by thyrotropin of thyroid organilication of circulating iodide. Endocrinology 78, 621-627. ROSENKRANTZ, H., HADIDIAN, Z., SEAY, H. and MASON, M. M. (1963). Dimethyl sulfoxide: its steroid solubility and endocrinologic and pharmacologic-toxicologic characteristics. Cancer Chemother. Rep. 31, 7-24. SILVERSTEIN, E. and BATES, R. W. (1961). Differences in thyroidal iodine concentration and T/S ratio among strains of mice and rats. Amer. J. Physiol. 200, 807-810. SMITH, E. R., HADIDIAN, Z. and MASON, M. M. (1967). The single-and repeated-dose toxicity of dimethyl sulfoxide. Ann. N. Y. Acad. Sci. 141, 96-109. STOUGHTON, R. (1965). Dimethyl sulfoxide induction of a steroid reservoir in human skin. Arch. Dermatol. 91, 657-660. STOUGHTON, R. and FRITSCH, B. W. (1964). Influence of dimethyl sulfoxide (DMSO) on human percutaneous absorption. Arch. Dermatol. 90, 512-517. VANDERLAAN, J. E. and VANDERLAAN, W. P. (1947). The iodide concentrating mechanism of the rat thyroid and its inhibition by thiocyanate. Endocrinology 40,403-416.
80
GOLDMAN
WARD, J. R., MILLER,M. L. and MARCUS,S. (1967).The effect of dimethyl sulfoxide on the
local Schwartzmanphenomenon.Ann. N. Y. Acad. Sci. 141,280-290. WOLFF,J. and CHAIKOFF,I. L. (1948).Plasmainorganic iodidesasa homeostaticregulator of thyroid function. J. Biol. Chem.174,555-564. WOLLMAN,S. H. and REED,F. E. (1959).Transport of radioiodide betweenthyroid glandand blood in mice and rats. Amer. J. Physiol. 196, 113-120.
APPLIEDPHARMACOLOGY-,
Failure of Dimethyl Thyroid Function
73-80(1973)
Sulfoxide (DMSO) in the Sprague-Dawley
to Alter Rat1
M. GOLDMAN Department of Biology, University of South Dakota, Vermillion, South Dakota 57069 Received February 23,1972
Failure of Dimethyl Sulfoxide (DMSO) to Alter Thyroid Function in the Rat. GOLDMAN, M. (1973). Toxicol. Appl. Pharmacol. 24, 73-80. Male Sprague-Dawleyrats 60 days and 1 year of age were injected with 63% or 85% dimethyl sulfoxide (DMSO) and severalphasesof thyroid function examined. Iodide transport was not affected by injection of either 63‘A or 85‘A DMSO. There was no significant differencebetween the saline-injectedand DMSO-injected groupsin the thyroid :serumradioiodide concentration ratios (T:S). The responseof the thyroid gland to the injection of an acute substantial iodide load was unaltered by prior injection of 85% DMSO. The inhibition in glandularbinding of iodide was similar in both saline-injectedand DMSO-injected groups as evidenced by the thyroidal accumulationof newly formed organiciodineand thyroidal concentration of iodide. Chromatographic analysesof thyroid pronase hydrolyzates in saline-injectedand DMSO-injected rats revealeda similar reduction in labelingof iodothyronines,an increasein the fraction of labeled iodide, anincreasein thyroidal labelingof MIT with consequentelevation in ratio of [r311]MIT:DIT. The radioiodine releaserate wasalsonot affected by injection of DMSO or dermal application of DMSO. Presentresults indicate that DMSO was not effective in influencing thyroid function in the rat. Numerous laboratory experimentation and clinical investigations have revealed that dimethyl sulfoxide (DMSO) has a wide spectrum of biological activity and yet is remarkably nontoxic (Rosenkrantz et al., 1963). It has been reported that DMSO increases the permeability of biological membranes (Jacob et al., 1964), is a good solvent for many steroids(Kligman, 1965; Stoughton, 1965),and augmentspercutaneous penetration of various drugs (Horita and Weber, 1964; Stoughton and Fritsch, 1964). DMSO is also radioprotective against ionizing radiation (Ashwood-Smith, 1961), protects cellsand tissuesagainst freezing damage(Lovelock and Bishop, 1959),prevents the inflammatory response (Ward et al., 1967), and relieves pain due to headache (Ogden, 1967). A recent report indicates that DMSO may have an inhibitory action on thyroid function (Hagemarm and Evans, 1968). These investigators reported that DMSO inhibited 1311uptake by mice thyroid glands both in vivo and in vitro. The inhibitory effect of DMSO on 1311uptake in vitro was simply removed by washing the thyroid glands in saline. Radioiodine uptake curves obtained over the period oftime during which thyroid glands were incubated in 15‘A DMSO appeared to parallel those uptake 1 Supportedby a grant from the University of SouthDakota’sGeneralResearch/NASAFund. Copyright All rights
0 1973 by Academic Press, Inc. of reproduction in any form reserved.
73
74
GOLDMAN
curves obtained when thyroid glands were incubated in thiocyanate (to block active transport of iodide) plus propylthiouracil (to block organification of iodide). Moreover, injection of 63 ‘A DMSO in mice resulted in a slight but significant depression in 1311 uptake 1 hr after injection. However, the depression in radioiodine uptake was not observable in these mice 6 hr later, presumably due to the fall in DMSO blood content. Consequently, Hagemann and Evans (1968) suggested that DMSO inhibited both active transport and organification of iodine by the thyroid gland. The present study reports our findings in investigating the action of DMSO on iodide transport and organification of iodine by the thyroid gland in the rat in an attempt to elucidate the mechanism of action by which DMSO inhibits thyroid function.
METHODS Two groups of male Sprague-Dawley rats obtained from our colony aged 60 days and 1 year old were used in this study. The animals were maintained on Purina Laboratory Chow and water ad libitum. DMSO’ was used at 2 dosages, 63% and 85% (v/v in physiological saline). All control animals were injected ip with 0.5 ml of physiological saline. Thyroid:serum radioiodide concentration ratios (T:S). Rats were injected ip with 10 mg methimazole3 in physiological saline in order to block organification of iodine, followed 1 hr later by an ip injection of 3-5 $i of carrier-free 13114.The administration of the radioiodide was preceded by a single ip injection of 0.5 ml DMSO in a concentration of 63 % or 85 % 5 min before the injection of the 1311.One hour later the thyroid glands were removed under light ether anesthesia, dissected, carefully weighed, and homogenized in 0.5 ml NaClaTris buffer, pH 8.5. An aliquot of homogenate was counted in a Nuclear Chicago well-type scintillation counter in order to calculate the amount of radioactivity in 1 g of thyroid tissue. Blood was obtained from the dorsal aorta after removal of the thyroid gland and an aliquot of serum counted to determine the radioactivity in 1 ml of serum. The thyroid-serum radioiodide concentration ratio (T:S) was calculated by determining the ratio of radioiodide per gram of thyroid to that of the radioiodide per milliliter of serum (Rosenberg et al., 1964). Distribution of 1311in hydrolyzed thyroidglands. Rats (60 days of age) received an ip injection of 0.5 ml 85 % DMSO which was followed 5 min later by an ip injection of 10 &i 1311together with 200 pg carrier iodide. The thyroid glands were removed 1 hr later, homogenized immediately in the NaCI*Tris buffer, pH 8.5, and hydrolyzed with pronase5. The radioactivity in an aliquot of homogenate was counted to determine the radioiodine uptake. The iodoaminoacids of the hydrolyzate were fractionated by paper chromatography in a collidine-3 N ammonia (3 : 1 v/v) solvent system and the position of the bands of radioactivity containing the iodinated amino acids were determined by autoradiography of the chromatograms. The radioactivity in these bands was determined after removal of the radioactive strips from the chromatogram and placement in glass vials following the procedure of Rosenberg et al. (1964). * Obtained from Mallinckrodt, St. Louis, Missouri. 3 I-Methyl-2-mercaptoimidazole, a gift from Eli Lilly and Company, Indianapolis, Indiana. 4 N.T obtained from Amersham/Searle, Arlington Heights, Illinois. .5 Streptomyces griseus protease, a product of Calbiochem, Los Angeles, California,
DMSO
AND
THYROID
FUNCTION
75
Distribution of 1311 in unhydrolyzed thyroid glands. Thyroid glands from another group of rats (60 days of age) similarly injected ip with 85 % DMSO followed 5 min later by an ip injection of 20 &i 131Ttogether with 200 ,ug iodide were homogenized in the NaClaTris buffer pH 8.5, and substantial aliquots of the unhydrolyzed glands were chromatographed using the same solvent system as above. Autoradiographs of the chromatograms localized the radioactivity into 2 bands: a band at the origin assumed to represent organic iodine and a band of iodide. The concentration of newly formed protein bound iodine (PB 12’1) and iodide 12’1 were calculated from the specific activity of the injected iodide, 1311uptake, and the fraction of 1311at the originaccording to the procedure of Rosenfeld and Rosenberg (I 966). Thyroid radioiodine release rate. Thyroidal 1311 release was measured by external neck counts taken over the thyroid region twice daily. The counting table had a top consisting of a lead plate 2 inches thick with an opening 1 inch in diameter directly above a scintillation probe which had a 2-inch NaI crystal. The probe was connected to a Nuclear Chicago scaler. Rats lightly anesthetized with ether were placed on the counting table and the neck positioned over the opening until the thyroid region was precisely above the opening over the scintillation probe. The initial thyroid count was taken 36 hr after an ip injection of 20 pCi carrier-free 1311and was considered as 100 )‘,; of thyroidal L31l. All subsequent counts were expressed as a percentage of the initial thyroidal uptake and plotted as a function of time on semilog paper. Blockage of thyroidal reaccumulation of 1311liberated from degradation of endogeneously labeled iodinated amino acids was not attempted. The procedure used was based on the method described by Albert (1951) and Reineke and Singh (1955). Hair from the lateral abdominal area was removed by plucking from 5 male rats 60 days of age, and during determination of the thyroidal 1311release rate 0.1 ml 1000; DMSO was dermally applied daily by means of a cahbrated medicine dropper. In a second experiment, after a straight line of more than 5 individual points was obtained for the thyroidal 1311release curve, 5 male rats 1 year old were injected ip daily with 0.5 ml 85 % DMSO. Analysis of the significance of differences between groups was done by means of Student’s t test. Ap value co.01 was considered statistically significant. RESULTS
Table 1 shows the data obtained in 2 experiments in which 2 different doses of DMSO were utilized in examining the effects of DMSO on the thyroid: serum radioiodide concentration ratio (T: S). The T: S values for the 2 control groups are typical of rats of that age group in our colony. Since 63 % DMSO did not affect the ability of the thyroid to trap iodide, the dose was consequently increased to 85 % DMSO. Again, the injected 85 % DMSO had no significant effect on iodide transport. The T: S ratios in the 2 experiments were not significantly different in the saline-injected and DMSUinjected rats. Thyroidal Organl$cation
of Iodide
The capacity to organify iodine can be studied by examining the thyroidal content of newly accumulated organic iodine after injection of an iodide load (Rosenfeld and
76
GOLDMAN
TABLE EFFECT OF 63 % DMSO
Group
No. of rats
Control DMSO (63 %) Control
10
DMSO
(83 %)
9 12 11
1
AND 85 % DMSO
Body weight (d
Age 60 days 60 days 1 year 1 year
ON T: S IODIDE IN MALE RATS’
201 * 8.3 183 rt 11.0 400 f 12.1 389 h 14.0
Thyroid weight @x4 13.0 zt 0.8 15.2 zt 0.7 17.7 i 1.1 16.1 zt 1.3
T:S 22.7 18.4 48.0 42.8
It rt k k
2.1b 2.6” 3.1 3.6”
a Rats received a single ip injection of 0.5 ml DMSO (63 % or 85 %) 5 min before ip injection of r3rI; thyroid glands removed 1 hr later and thyroid radioiodide concentration/serum radioiodide concentration ratio calculated. b Mean value * SE. c Not significantly different from controls.
Rosenberg, 1966). Table 2 shows the effect of 200 pg of iodide in causing a reduction in organic binding of iodine by the thyroid gland (Wolff and Chaikoff, 1948) in salineinjected and 85% DMSO-injected rats. The data presented in Table 2 show that the 1311uptake, the fraction of 1311organically bound, the accumulation of newly formed organic iodine, and the concentration of accumulated inorganic iodide were not significantly different in the thyroid glands of saline-injected and DMSO-injected rats subjected to an acute iodide load. Alterations in intrathyroidal iodine metabolism which occurred in response to the injection of 200 pg iodide were not influenced by DMSO. TABLE
2
EFFECT OF 85 % DMSO ON ORGANIFICATION OF AN INJECTED IODIDE LOAD (200 pg) IN MALE RATS 60 DAYS OLDO Organic
1311Uptake Group
No. of rats
Control DMSO (85 %)
12 12
1311
‘A injected
organically
(dosehw)
bound
0.01 f 0 0.01 i 0
11.1 f 3.8
12.7 zt 3.0
Iodine newly formed (wims) 2.30 zk 0.52 1.59 rt 0.59
Inorganic iodide accumulated Cx/mg) 15.8 4 1.7b 13.4 f 1.7
a Rats received a single ip injection of 0.5 ml 85 % DMSO 5 min before ip injection of 20 &i r3’I plus 200 pg carrier iodide; thyroid glands removed 1 hr later, homogenized, and chromatographed. b Mean value f SE.
Radioiodine Distribution in Componentsof Pronase Thyroid Hydrolyzates
’ Chromatographic analyses of thyroid hydrolyzates are shown in Table 3. A marked reduction in percentage of 1311in iodothyronines (T3 + T4) and iodotyrosines (MIT and DIT) is observable in both saline-injected and 85 ‘A DMSO-injected rats. The large increase in the fraction of labeled iodide occurred in both groups of rats and again there was no significant difference in percentage of thyroidal label in this component. These results are consistent with the data observed in Table 2, showing the marked
DMSO
AND
THYROID
77
FUNCTION
impairment in thyroidal capacity to bind iodine organically after injection of 200 pg iodide. The fraction of thyroidal label in MIT is considerably higher than in DIT in both groups of rats and results in a significant increase in MIT: DIT ratio (Table 3). The data shown in Tables 2 and 3 are evidence that the Wolff-Chaikoff effect (inhibition of iodothyronine synthesis) occurred in both saline-treated and DMSO-treated rats in response to the injected iodide load without any influence of DMSO on the iodide block in organification of iodine. TABLE DISTRIBUTION OF RATS 60 DAYS
Group Control DMSO
OF 1311(‘A) IN CHROMATOGRAMS OF AGE INJECTED WITH 85 ‘A IODIDP
No. of rats 10
(85 %)
10
3 OF THYROID HYDROLYZATES AND 200 pg OF CARRIER
DMSO
Origin
MIT
DIT
0.7 iz 0.2 0.5 ~0.2
3.0 50.8 6.7 * 1.6
1.5 i0.6 3.6 zk 1.7
Tx+Tb 1.5 ho.6 1.7 ztO.4
1311 93.4 -+ 1.8 87.5 312.8
MIT:DIT 2.0 ~~0.5~ 1.0 $I 0.5
” Rats received a single ip injection of 0.5 ml 85 % DMSO 5 min before ip injection of 20 &i 13’1 plus 200 pg carrier iodide; thyroid glands removed 1 hr later, homogenized, hydrolyzed with pronase and chromatographed. MIT; monoiodotyrosine; DIT, Diiodotyrosine; TA, triiodothyronine; Tj, thyroxine. h Mean value -I SE.
Tllyroidal Radioiodine Release
Figure 1 shows the effect of DMSO on the thyroidal secretion rate. Daily in,jections of 0.5 ml 85 y{ DMSO did not alter the rate of 1311secretion from the thyroid gland. Dermal application of concentrated DMSO also did not alter 1311 release from the
0
I
I
I
I
30
60
90
120
I
150
HOURS FIG. 1. Effect of DMSO on thyroidal radioiodine release, Male rats 60 days of age were injected ip daily with 0.5 ml 85 % DMSO where noted (4). The curve represents the means f SE of determinations made on 5 rats.
78
GOLDMAN
thyroid. Since there were no changes in slope of the thyroid 1311release rates when DMSO was injected or dermally applied, only the thyroidal radioiodine release curve for DMSO injected rats is shown. The biological half-life determined graphically was 3.3 days for both control and DMSO-treated rats. DISCUSSION The active transport of iodide, the first step in the metabolism of iodine, can be examined independently and separately of subsequent steps in thyroid hormone biosynthesis. The efficiency of the thyroid gland in iodide transport is measured experimentally as the thyroid : serum radioiodide concentration ratio (T: S) and expresses the concentration gradient for radioiodide which the thyroid maintains against the plasma (Vanderlaan and Vanderlaan, 1947). The data presented in this study of the ineffectiveness of DMSO in depressing the active transport of iodide are at variance with the suggestion of Hagemann and Evans (1968) that DMSO inhibits this phase of thyroid function. DMSO given at 2 different doses did not inhibit iodide transport by the thyroid gland. Synthesis of the thyroid hormones, thyroxine and triiodothyronine, results from the coupling of iodotyrosines subsequent to organic binding of the trapped iodide. Hagemann and Evans (1968) claim that DMSO also inhibits organification of iodine by the thyroid gland. While administration of small doses of carrier iodide leads to an increase in thyroidal organic iodinations, large doses of iodide result in a depression of organic binding of iodide. This temporary inhibitory effect on organification of iodide and consequent depression in synthesis of thyroid hormones as a result of a large dose of iodide has been termed the Wolff-Chaikoff effect and has been shown to be related to the increased concentration of intrathyroidal iodide (Raben, 1949). Consequently, an estimate of the capacity of the thyroid gland to organify iodine can be obtained by examining the glandular content of newly formed organic iodine after injection of a substantial iodide load (Deodhar and Rosenberg, 1969). Our results do not confirm Hagemann and Evans’ (1968) suggestion that DMSO inhibits thyroidal organification of iodide. The organic iodine and iodide newly accumulated in the thyroid glands of control and DMSO injected rats were similar after injection of the 200 pg iodide. The inhibition in glandular binding of iodide by the injected iodide was of similar magnitude in both control and DMSO-treated rats and was not influenced by injection of DMSO. The marked depression in thyroidal labeling of iodothyronines, iodotyrosines and the increased fraction of 1311-labeled iodide were not significantly different in the 2 groups. Moreover, the increase in labeling of the MIT component was reflected as a significant increase in MIT:DIT ratio in both groups. Galton and Pitt-Rivers (1959) have pointed out that the MIT : DIT ratio is very high when organic binding of iodide is most depressed. No inhibition in thyroidal secretion rate was observed after dermal application of an injection of 85 7; DMSO. In contrast to the demonstration of an inhibitory effect of DMSO on thyroid function in the mouse reported by Hagemann and Evans (1968), the present investigation does not reveal any demonstrable effect of DMSO on thyroid function in the rat. Conse-
DMSO AND THYROID FUNCTION
79
quently, we have not been able to confirm the mechanism of action for DMSO inhibition of thyroid function suggestedby theseinvestigators. Although a remarkable lack of speciesvariation in responseto DMSO has been reported (Smith et al., 1967), our results do not deny the existence of possibledifferences in metabolism of DMSO in the rat and mousewhich could resolve the disparity in findings reported here with those of Hagemann and Evans (1968), particularly in view of the reports of speciesdifferences in thyroid function in the rat and mouse (Wollman and Reed, 1959; Silverstein and Bates, 1961). REFERENCES ALBERT,
A. (1951).The in vivo determinationof the biological decayof thyroidal radioiodine.
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M. J. (1961).The radioprotective action of dimethyl sulphoxideand various other sulphoxides.Int. J. Radiat. Biol. 3, 41-48. DEODHAR, S. D. and ROSENBERG, I. N. (1969). Effects of actinomycin D on the thyroid. ASHWOOD-SMITH,
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GOLDMAN
WARD, J. R., MILLER,M. L. and MARCUS,S. (1967).The effect of dimethyl sulfoxide on the
local Schwartzmanphenomenon.Ann. N. Y. Acad. Sci. 141,280-290. WOLFF,J. and CHAIKOFF,I. L. (1948).Plasmainorganic iodidesasa homeostaticregulator of thyroid function. J. Biol. Chem.174,555-564. WOLLMAN,S. H. and REED,F. E. (1959).Transport of radioiodide betweenthyroid glandand blood in mice and rats. Amer. J. Physiol. 196, 113-120.